The N-terminal tails of histone proteins are heavily decorated by a plethora of covalent modifications including methylation, acetylation, phosphorylation, sumoylation and ubiquitylation. These modifications work together to generate appropriate chromatin templates for a host of important nuclear events, including transcription, DNA replication, recombination and repair. A defect in the DNA damage signaling and repair machinery can lead to the development of cancer, and therefore understanding the mechanisms that control this pathway has important health implications. In recent years, although significant progress has been made in our understanding of histone modifications in transcriptional regulation, much less is known about how these modifications impact the DNA damage response (DDR) and the precise molecular mechanisms by which histone- modifying enzymes regulate the DDR pathway. In the past funding period, we discovered that the histone demethylase LSD1, which thus far is thought to function only as a transcriptional regulator, also plays an evolutionarily conserved role in the DDR. Loss of LSD1 does not affect the expression of repair genes;furthermore, chromatin immunoprecipitation places LSD1 at DNA damage sites, suggesting a direct involvement of LSD1 in this process. Importantly, loss of LSD1 results in impaired histone H2A/H2A.X ubiquitylation, with subsequent loss of 53BP1 recruitment, a molecule important for DNA damage repair. In this application, we propose to test our hypothesis that a novel crosstalk exists between histone H3K4 demethylation and H2A/H2A.X ubiquitylation important for the DDR by identifying molecular mechanisms by which LSD1 regulates histone H2A/H2A.X ubiquitylation. Given that LSD1 mediates demethylation of mono- and di-methylated histone H3K4, we will also investigate whether H3K4 methylation dynamics is generally important for the DDR by determining whether demethylases for the tri-methylated H3K4 as well as a host of H3K4 methyltransferases also play a role in the DDR. Lastly we will begin to address the question of whether there is a specific constellation of histone modifications at DNA damage sites that induce a distinct chromatin domain for DNA repair, by delineating the histone modification landscape and the responsible enzymes at DNA damage sites. We anticipate that new paradigms to emerge from the proposed studies, significantly impacting our understanding of epigenetic regulation in the DDR. PUBLIC HEALTH RELEVANCE: This application proposes to study the histone-modifying enzyme LSD1 in the DNA damage response pathway. Findings will significantly improve our understanding of how epigenetic regulators are involved in the regulation of genomic stability, which may impact the approaches used in epigenetic therapy.